1. Field of the Invention
The present invention relates to a baculovirus vector which is suitable for use in gene therapy, etc., a method of constructing the baculovirus vector and a method of gene transfer using the baculovirus vector.
2. Description of the Related Art
Ten years have passed since clinical studies of gene therapy started. Until now, a satisfactory outcome has not been achieved; however, this therapy, without doubt, will be at the center of advanced medical treatment in this century. Whether or not gene therapy will successfully advance depends on the development of gene transfer vectors which enable introduction of genes into target cells safely and efficiently and which have a large integration capacity.
Retroviruses, adenoviruses, adeno-associated viruses, etc. have been the main vectors used in gene therapy. However, there have been safety problems, such as the emergence of self-propagating virus and the activation of cancer genes due to random integration of genetic material, and satisfactory gene transfer efficiency and improvement of specific gene transfer methods has not been achieved. Further, there have been problems such as cytotoxicity, induction of undesirable immune reaction, and inactivation due to neutralizing antibodies.
Baculovirus, an insect virus, was thought to infect only insects until recently and had been utilized as a large-scale expression system of transgenes in insect cells.
Baculoviruses are entomopathogenic viruses which infect insects such as lepidopteran, hymenopteran and diptera and have circular double-stranded DNA. Of the viruses, a group of viruses called Nuclear Polyhedorosis Virus (NPV) produce a large amount of inclusion bodies called polyhedrins in the nucleus of the infected cell during the late phase of infection. The amount of the inclusion body reaches as much as 40 to 50% of the total cell protein and the inclusion body includes a large number of viral particles (FIG. 1). Thus, even if the host is killed by infection, viruses in this polyhedrin inclusion body are protected from inactivation by, for example, ultraviolet radiation and heat, thus enabling the viruses to remain infectious for a long time (FIG. 2).
Thus, polyhedrins are essential for viruses to survive in nature, but they are not required for propagation of viruses in vitro. Therefore, if a foreign gene intended to be expressed is introduced in place of a polyhedrin gene, infection and propagation of the virus will occur and a large amount of foreign gene products will be produced without any difficulties. Until now, two viruses, Autographa californica NPV (AcNPV) and Bombyx mori NPV (BmNPV) of silkworm, have been used as vectors, but in most cases, expression is carried out using AcNPV.
The present inventors have previously succeeded in the development of the vector pAcYM1 which has the highest expression efficiency in the world among baculovirus vectors (e.g. Matsuura, Y., Possee, R. D., Overton, H. A., and Bishop, D. H. L. (1987), “Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins,” J. Gen. Virol. 68, 1233-1250). They developed the expression of hepatitis C virus structural protein using the vector and succeeded in the development of an early stage antibody diagnosis system using it as an antigen. Due to the introduction of this diagnostic system, the incidence of post-tranfusion hepatitis C is almost conquered in Japan.
In recent years, gene transfer into mammalian cells using baculovirus has been reported (e.g. Hofmann, C., Sandig, V., Jennings, G., Rudolph, M., Schlag, P., and Strauss, M. (1995), “Efficient gene transfer into human hepatocytes by baculovirus vectors,” Proc. Natl. Acad. Sci. USA 92, 10099-10103, and Boyce, F. M., and Bucher, N. L. R. (1996), “Baculovirus-mediated gene transfer into mammalian cells,” Proc. Natl. Acad. Sci. USA 93, 2348-2352). At first, it was thought gene transfer using baculovirus was specific to hepatocytes; however, the present inventors demonstrated the capability of gene transfer into a wide variety of animal cells (e.g. Shoji I., Aizaki H., Tani H., Ishii K., Chiba T., Saito I., Miyamura T., and Matsuura Y. (1997), “Efficient gene transfer into various mammalian cells including non-hepatic cells by baculovirus vectors,” J. Gen. Virol. 78, 2657-2664).
In this way, it has been demonstrated that baculovirus vectors infect a wide variety of mammalian cells and can express foreign genes efficiently without replication. Therefore, the baculovirus vectors have attracted attention as a potential gene therapy vector.
Baculovirus vectors have extraordinary properties as a gene transfer vector into humans for the following reasons: 1) the virus gene is as large as 130 kbp, which allows insertion of large (<15 kbp) foreign genes, 2) since the virus gene is not expressed in mammalian cells, it hardly causes cytotoxicity and a harmful immune response is not induced, 3) recombinant virus can be prepared in a short time, 4) there exists no neutralizing antibody against baculovirus in the human, etc.
In order to improve the infection efficiency in mammalian cells, an attempt has been made to provide other virus envelope proteins such as vesicular stomatitis virus G protein in baculovirus (e.g. Tani, H., Nishijima, M., Ushijima, H., Miyamura, T., and Matsuura, Y. (2001), “Characterization of cell-surface determinants important for baculovirus infection,” Virology 279, 343-353) (FIG. 3 left). This remarkably improved the gene transfer efficiency into mammalian cells.
Further, another attempt has been made to completely replace the baculovirus gp64 protein with the envelope protein of another virus, and it is reported that the recombinant virus can replicate in insect cells when the gp64 protein is replaced by envelope proteins of closely related virus or vesicular stomatitis virus G protein, (e.g. Mangor J. T., Monsam S. A., Johnson C. M., and Blissard G. W. (2001), “A gp64-null baculovirus pseudotyped with vesicular stomatitis virus G protein.” J. Virol. 75, 2544-2556).
However, in these conventional methods, viruses are required to be infectious to insect cells to obtain a high titer of stock virus. Thus in these prior methods, proteins having no infectivity to insect cells could not be selected.
Therefore, it is impossible to make infection of these vectors specific and a baculovirus vector capable of conferring specificity has not been reported. The development of a baculovirus vector which has the above-mentioned excellent properties and which is capable of conferring the desired infectiousness was strongly desired.